Protosequences in human cortical organoids model intrinsic states in the developing cortex.

Neuronal firing sequences are thought to be the basic building blocks of neural coding and information broadcasting within the brain. However, when sequences emerge during neurodevelopment remains unknown. We demonstrate that structured firing sequences are present in spontaneous activity of human brain organoids and ex vivo neonatal brain slices from the murine somatosensory cortex. We observed a balance between temporally rigid and flexible firing patterns that are emergent phenomena in human brain organoids and early postnatal murine somatosensory cortex, but not in primary dissociated cortical cultures. Our findings suggest that temporal sequences do not arise in an experience-dependent manner, but are rather constrained by an innate preconfigured architecture established during neurogenesis. These findings highlight the potential for brain organoids to further explore how exogenous inputs can be used to refine neuronal circuits and enable new studies into the genetic mechanisms that govern assembly of functional circuitry during early human brain development.

Functional neuronal circuitry and oscillatory dynamics in human brain organoids.

Human brain organoids replicate much of the cellular diversity and developmental anatomy of the human brain. However, the physiology of neuronal circuits within organoids remains under-explored. With high-density CMOS microelectrode arrays and shank electrodes, we captured spontaneous extracellular activity from brain organoids derived from human induced pluripotent stem cells. We inferred functional connectivity from spike timing, revealing a large number of weak connections within a skeleton of significantly fewer strong connections. A benzodiazepine increased the uniformity of firing patterns and decreased the relative fraction of weakly connected edges. Our analysis of the local field potential demonstrate that brain organoids contain neuronal assemblies of sufficient size and functional connectivity to co-activate and generate field potentials from their collective transmembrane currents that phase-lock to spiking activity. These results point to the potential of brain organoids for the study of neuropsychiatric diseases, drug action, and the effects of external stimuli upon neuronal networks.

Dynamic Phase Extraction: Applications in Pulse Rate Variability.

Pulse rate variability is a physiological parameter that has been extensively studied and correlated with many physical ailments. However, the phase relationship between inter-beat interval, IBI, and breathing has very rarely been studied. Develop a technique by which the phase relationship between IBI and breathing can be accurately and efficiently extracted from photoplethysmography (PPG) data. A program based on Lock-in Amplifier technology was written in Python to implement a novel technique, Dynamic Phase Extraction. It was tested using a breath pacer and a PPG sensor on 6 subjects who followed a breath pacer at varied breathing rates. The data were then analyzed using both traditional methods and the novel technique (Dynamic Phase Extraction) utilizing a breath pacer. Pulse data was extracted using a PPG sensor. Dynamic Phase Extraction (DPE) gave the magnitudes of the variation in IBI associated with breathing [Formula: see text] measured with photoplethysmography during paced breathing (with premature ventricular contractions, abnormal arrhythmias, and other artifacts edited out). [Formula: see text] correlated well with two standard measures of pulse rate variability: the Standard Deviation of the inter-beat interval (SDNN) (ρ = 0.911) and with the integrated value of the Power Spectral Density between 0.04 and 0.15 Hz (Low Frequency Power or LF Power) (ρ = 0.885). These correlations were comparable to the correlation between the SDNN and the LF Power (ρ = 0.877). In addition to the magnitude [Formula: see text], Dynamic Phase Extraction also gave the phase between the breath pacer and the changes in the inter-beat interval (IBI) due to respiratory sinus arrythmia (RSA), and correlated well with the phase extracted using a Fourier transform (ρ = 0.857). Dynamic Phase Extraction can extract both the phase between the breath pacer and the changes in IBI due to the respiratory sinus arrhythmia component of pulse rate variability ([Formula: see text], but is limited by needing a breath pacer.

Extracellular detection of neuronal coupling.

We developed a method to non-invasively detect synaptic relationships among neurons from in vitro networks. Our method uses microelectrode arrays on which neurons are cultured and from which propagation of extracellular action potentials (eAPs) in single axons are recorded at multiple electrodes. Detecting eAP propagation bypasses ambiguity introduced by spike sorting. Our methods identify short latency spiking relationships between neurons with properties expected of synaptically coupled neurons, namely they were recapitulated by direct stimulation and were sensitive to changing the number of active synaptic sites. Our methods enabled us to assemble a functional subset of neuronal connectivity in our cultures.

Significant correlation of bone material strength index as measured by the OsteoProbe with Vickers and Rockwell hardness.

The bone material strength index (BMSi), as measured by the OsteoProbe, is significantly correlated with Vickers hardness and Rockwell (RW) hardness measurements on conventional materials. The Vickers and RW measurements were carried out according to American Society for Testing and Materials standard test methods, and OsteoProbe measurements followed published standardized testing methods. The correlations between the BMSi and RW hardness, r = 0.93, and between the BMSi and Vickers hardness, r = 0.94, are comparable with the correlation between RW and Vickers hardness, r = 0.87. The correlation between the BMSi and RW is significant at p < 0.01, and the correlation between the BMSi and Vickers hardness is significant at p < 0.01. These results show that the indentation measurement performed by the OsteoProbe may be considered as a type of hardness measurement comparable to widely used conventional methods, with specific applications targeted by its portable and narrow design.

Non-contact monitoring of extra-cellular field potentials with a multi-electrode array.

Developing tools to enable non-invasive, high-throughput electrophysiology measurements of large functional-networks of electrogenic cells used as in vitro disease models for the heart and brain remains an outstanding challenge for preclinical drug discovery, where failures are costly and can prove to be fatal during clinical trials. Here we demonstrate, for the first time, that it is possible to perform non-contact monitoring of extra-cellular field potentials with a multi-electrode array (MEA). To do this preliminary demonstration we built a prototype with a custom mechanical stage to micro-position cells grown on conventional glass coverslips over the recording surface of a MEA sensor. The prototype can monitor extra-cellular fields generated by multi-cellular networks in a non-contact configuration, enabling a single MEA sensor to probe different cultures in succession, without fouling or degrading its sensitive electronic surface. This first demonstration with easy to culture cardiomyocyte cells and a prototype device points to the exciting possibility for instrument development leading to more efficient and cost-effective drug screening paradigms for cardiovascular and neurological diseases.

Action potential propagation recorded from single axonal arbors using multielectrode arrays.

We report the presence of co-occurring extracellular action potentials (eAPs) from cultured mouse hippocampal neurons among groups of planar electrodes on multielectrode arrays (MEAs). The invariant sequences of eAPs among coactive electrode groups, repeated co-occurrences, and short interelectrode latencies are consistent with action potential propagation in unmyelinated axons. Repeated eAP codetection by multiple electrodes was widespread in all our data records. Codetection of eAPs confirms they result from the same neuron and allows these eAPs to be isolated from all other spikes independently of spike sorting algorithms. We averaged co-occurring events and revealed additional electrodes with eAPs that would otherwise be below detection threshold. We used these eAP cohorts to explore the temperature sensitivity of action potential propagation and the relationship between voltage-gated sodium channel density and propagation velocity. The sequence of eAPs among coactive electrodes "fingerprints" neurons giving rise to these events and identifies them within neuronal ensembles. We used this property and the noninvasive nature of extracellular recording to monitor changes in excitability at multiple points in single axonal arbors simultaneously over several hours, demonstrating independence of axonal segments. Over several weeks, we recorded changes in interelectrode propagation latencies and ongoing changes in excitability in different regions of single axonal arbors. Our work illustrates how repeated eAP co-occurrences can be used to extract physiological data from single axons with low-density MEAs. However, repeated eAP co-occurrences lead to oversampling spikes from single neurons and thus can confound traditional spike-train analysis. NEW & NOTEWORTHY We studied action potential propagation in single axons using low-density multielectrode arrays. We unambiguously identified the neuronal sources of propagating action potentials and recorded extracellular action potentials from several positions within single axonal arbors. We found a surprisingly high density of axonal voltage-gated sodium channels responsible for a high propagation safety factor. Our experiments also demonstrate that excitability in different segments of single axons is regulated independently on timescales from hours to weeks.

MEA Viewer: A high-performance interactive application for visualizing electrophysiological data.

Action potentials can be recorded extracellularly from hundreds of neurons simultaneously with multi-electrode arrays. These can typically have as many as 120 or more electrodes. The brief duration of action potentials requires a high sampling frequency to reliably capture each waveform. The resulting raw data files are therefore large and difficult to visualize with traditional plotting tools. Common approaches to deal with the difficulties of data display, such as extracting spike times and performing spike train analysis, are useful in many contexts but they also significantly reduce data dimensionality. The use of tools which minimize data processing enable the development of heuristic perspective of experimental results. Here we introduce MEA Viewer, a high-performance open source application for the direct visualization of multi-channel electrophysiological data. MEA Viewer includes several high-performance visualizations, including an easily navigable overview of recorded extracellular action potentials from all data channels overlaid with spike timestamp data and an interactive raster plot. MEA Viewer can also display the two dimensional extent of action potential propagation in single neurons by signal averaging extracellular action potentials (eAPs) from single neurons detected on multiple electrodes. This view extracts and displays eAP timing information and eAP waveforms that are otherwise below the spike detection threshold. This entirely new method of using MEAs opens up novel research applications for medium density arrays. MEA Viewer is licensed under the General Public License version 3, GPLv3, and is available at http://github.com/dbridges/mea-tools.

Effect of various testing conditions on results for a handheld reference point indentation instrument in horses.

OBJECTIVE: To compare results obtained with a handheld reference point indentation instrument for bone material strength index (BMSi) measurements in the equine third metacarpal bone for various testing conditions. SAMPLE: 24 third metacarpal bones. PROCEDURES: Third metacarpal bones from both forelimbs of 12 horses were obtained. The dorsal surface of each bone was divided into 6 testing regions. In vivo and ex vivo measurements of BMSi were obtained through the skin and on exposed bone, respectively, to determine effects of each testing condition. Difference plots were used to assess agreement between BMSi obtained for various conditions. Linear regression analysis was used to assess effects of age, sex, and body weight on BMSi. A mixed-model ANOVA was used to assess effects of age, sex, limb, bone region, and testing condition on BMSi values. RESULTS: Indentation measurements were performed on standing sedated and recumbent anesthetized horses and on cadaveric bone. Regional differences in BMSi values were detected in adult horses. A significant linear relationship (r(2) = 0.71) was found between body weight and BMSi values. There was no difference between in vivo and ex vivo BMSi values. A small constant bias was detected between BMSi obtained through the skin, compared with values obtained directly on bone. CONCLUSIONS AND CLINICAL RELEVANCE: Reference point indentation can be used for in vivo assessment of the resistance of bone tissue to microfracture in horses. Testing through the skin should account for a small constant bias, compared with results for testing directly on exposed bone.

Bone Tissue Properties Measurement by Reference Point Indentation in Glucocorticoid-Induced Osteoporosis.

Glucocorticoids, widely used in inflammatory disorders, rapidly increase bone fragility and, therefore, fracture risk. However, common bone densitometry measurements are not sensitive enough to detect these changes. Moreover, densitometry only partially recognizes treatment-induced fracture reductions in osteoporosis. Here, we tested whether the reference point indentation technique could detect bone tissue property changes early after glucocorticoid treatment initiation. After initial laboratory and bone density measurements, patients were allocated into groups receiving calcium + vitamin D (Ca+D) supplements or anti-osteoporotic drugs (risedronate, denosumab, teriparatide). Reference point indentation was performed on the cortical bone layer of the tibia by a handheld device measuring bone material strength index (BMSi). Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA). Although Ca+D-treated patients exhibited substantial and significant deterioration, risedronate-treated patients exhibited no significant change, and both denosumab- and teriparatide-treated participants exhibited significantly improved BMSi 7 weeks after initial treatment compared with baseline; these trends remained stable for 20 weeks. In contrast, no densitometry changes were observed during this study period. In conclusion, our study is the first to our knowledge to demonstrate that reference point indentation is sensitive enough to reflect changes in cortical bone indentation after treatment with osteoporosis therapies in patients newly exposed to glucocorticoids.

A new ion sensing deep atomic force microscope.

Here we describe a new deep atomic force microscope (AFM) capable of ion sensing. A novel probe assembly incorporates a micropipette that can be used both for sensing ion currents and as the tip for AFM imaging. The key advance of this instrument over previous ion sensing AFMs is that it uses conventional micropipettes in a novel suspension system. This paper focuses on sensing the ion current passively while using force feedback for the operation of the AFM in contact mode. Two images are obtained simultaneously: (1) an AFM topography image and (2) an ion current image. As an example, two images of a MEMS device with a microchannel show peaks in the ion current as the pipette tip goes over the edges of the channel. This ion sensing AFM can also be used in other modes including tapping mode with force feedback as well as in non-contact mode by utilizing the ion current for feedback, as in scanning ion conductance microscopy. The instrument is gentle enough to be used on some biological samples such as plant leaves.

Applications of a New Handheld Reference Point Indentation Instrument Measuring Bone Material Strength.

A novel, hand-held Reference Point Indentation (RPI) instrument, measures how well the bone of living patients and large animals resists indentation. The results presented here are reported in terms of Bone Material Strength, which is a normalized measure of how well the bone resists indentation, and is inversely related to the indentation distance into the bone. We present examples of the instrument's use in: (1) laboratory experiments on bone, including experiments through a layer of soft tissue, (2) three human clinical trials, two ongoing in Barcelona and at the Mayo Clinic, and one completed in Portland, OR, and (3) two ongoing horse clinical trials, one at Purdue University and another at Alamo Pintado Stables in California. The instrument is capable of measuring consistent values when testing through soft tissue such as skin and periosteum, and does so handheld, an improvement over previous Reference Point Indentation instruments. Measurements conducted on horses showed reproducible results when testing the horse through tissue or on bare bone. In the human clinical trials, reasonable and consistent values were obtained, suggesting the Osteoprobe® is capable of measuring Bone Material Strength in vivo, but larger studies are needed to determine the efficacy of the instrument's use in medical diagnosis.

Microindentation for in vivo measurement of bone tissue material properties in atypical femoral fracture patients and controls.

Atypical femoral fractures (AFF) associated with long-term bisphosphonates (LTB) are a growing concern. Their etiology is unknown, but bone material properties might be deteriorated. In an AFF series, we analyzed the bone material properties by microindentation. Four groups of patients were included: 6 AFF, 38 typical osteoporotic fractures, 6 LTB, and 20 controls without fracture. Neither typical osteoporotic fractures nor controls have received any antiosteoporotic medication. A general laboratory workup, bone densitometry by dual-energy X-ray absorptiometry (DXA), and microindentation testing at the tibia were done in all patients. Total indentation distance (Total ID), indentation distance increase (IDI), and creep indentation distance (Creep ID) were measured (microns). Age-adjusted analysis of covariance (ANCOVA) was used for comparisons. Controls were significantly younger than fracture groups. Bisphosphonate exposure was on average 5.5 years (range 5 to 12 years) for the AFF and 5.4 years (range 5 to 8 years) for the LTB groups. Total ID (microns) showed better material properties (lower Total ID) for controls 36 (± 6; mean ± SD) than for AFF 46 (± 4) and for typical femoral fractures 47 (± 13), respectively. Patients on LTB showed values between controls and fractures, 38 (± 4), although not significantly different from any of the other three groups. IDI values showed a similar pattern 13 (± 2), 16 (± 6), 19 (± 3), and 18 (± 5). After adjusting by age, significant differences were seen between controls and typical (p < 0.001) and atypical fractures (p = 0.03) for Total ID and for IDI (p < 0.001 and p < 0.05, respectively). There were no differences in Creep ID between groups. Our data suggest that patients with AFF have a deep deterioration in bone material properties at a tissue level similar to that for the osteoporotic fracture group. The LTB group shows levels that are in between controls and both type of fractures, although not statistically different. These results suggest that bisphosphonate therapy probably does not put the majority of patients at risk for AFF.

A new device for performing reference point indentation without a reference probe.

Here we describe a novel, hand-held reference point indentation (RPI), instrument that is designed for clinical measurements of bone material properties in living patients. This instrument differs from previous RPI instruments in that it requires neither a reference probe nor removal of the periosteum that covers the bone, thus significantly simplifying its use in patient testing. After describing the instrument, we discuss five guidelines for optimal and reproducible results. These are: (1) the angle between the normal to the surface and the axis of the instrument should be less than 10°, (2) the compression of the main spring to trigger the device must be performed slowly (>1 s), (3) the probe tip should be sharper than 10 µm; however, a normalized parameter with a calibration phantom can correct for dull tips up to a 100 µm radius, (4) the ambient room temperature should be between 4 °C and 37 °C, and (5) the effective mass of the bone or material under test must exceed 1 kg, or if under 1 kg, the specimen should be securely anchored in a fixation device with sufficient mass (which is not a requirement of previous RPI instruments). Our experience is that a person can be trained with these guidelines in about 5 min and thereafter obtain accurate and reproducible results. The portability, ease of use, and minimal training make this instrument suitable to measure bone material properties in a clinical setting.

Local tissue properties of human osteoarthritic cartilage correlate with magnetic resonance T(1) rho relaxation times.

The objective of this study is to examine the local relationship between T(1ρ) relaxation times and the mechanical behavior of human osteoarthritic articular cartilage using high-resolution magnetic resonance imaging (MRI) and local in situ microindentation. Seven human tibial plateaus were obtained from patients who underwent total knee arthroplasty due to severe osteoarthritis (OA). Three to six sites were selected from each sample for visual classification using the ICRS Outerbridge scale (a total of 36 sites). Samples were imaged by MR, and the local distribution of T(1ρ) relaxation times were obtained at these selected sites. The elastic and viscoelastic characteristics of the tissue were quantified nondestructively using dynamic microindentation to measure peak dynamic modulus, energy dissipation, and phase angle. Measured Outerbridge scores, MR T(1ρ) relaxation times, and mechanical properties were highly heterogeneous across each cartilage surface. Site-specific measures of T(1ρ) relaxation times correlated significantly with the phase angle (p < 0.001; R = 0.908), a viscoelastic mechanical behavior of the cartilage. The novel combination of high-resolution MR imaging and microindentation allows the investigation of the local relationship between quantitative MRI and biomechanical properties in highly heterogeneous OA cartilage. These findings suggest that MRI T(1ρ) can provide a functional assessment of articular cartilage.

Microindentation for in vivo measurement of bone tissue mechanical properties in humans.

Bone tissue mechanical properties are deemed a key component of bone strength, but their assessment requires invasive procedures. Here we validate a new instrument, a reference point indentation (RPI) instrument, for measuring these tissue properties in vivo. The RPI instrument performs bone microindentation testing (BMT) by inserting a probe assembly through the skin covering the tibia and, after displacing periosteum, applying 20 indentation cycles at 2 Hz each with a maximum force of 11 N. We assessed 27 women with osteoporosis-related fractures and 8 controls of comparable ages. Measured total indentation distance (46.0 +/- 14 versus 31.7 +/- 3.3 microm, p = .008) and indentation distance increase (18.1 +/- 5.6 versus 12.3 +/- 2.9 microm, p = .008) were significantly greater in fracture patients than in controls. Areas under the receiver operating characteristic (ROC) curve for the two measurements were 93.1% (95% confidence interval [CI] 83.1-100) and 90.3% (95% CI 73.2-100), respectively. Interobserver coefficient of variation ranged from 8.7% to 15.5%, and the procedure was well tolerated. In a separate study of cadaveric human bone samples (n = 5), crack growth toughness and indentation distance increase correlated (r = -0.9036, p = .018), and scanning electron microscope images of cracks induced by indentation and by experimental fractures were similar. We conclude that BMT, by inducing microscopic fractures, directly measures bone mechanical properties at the tissue level. The technique is feasible for use in clinics with good reproducibility. It discriminates precisely between patients with and without fragility fracture and may provide clinicians and researchers with a direct in vivo measurement of bone tissue resistance to fracture.

Mechanical profiling of intervertebral discs.

Despite recent advances in imaging diagnostic technology and additional treatment options our ability to prevent or inhibit discogenic back pain has not drastically improved. The challenge of linking early degenerative patterns to dysfunction and pain remains. Using a novel material testing device designated the tissue diagnostic instrument (TDI) we measured the local stiffness and strain energy absorption in the radial direction of 13 intact intervertebral discs; effectively generating a mechanical profile of each disc. Prior to measuring mechanical properties, an MR image was taken of each spine segment and the discs were radiologically scored according to the Pfirrmann scale. After testing, a sagittal portion of each L1-L2 disc was excised from each of four spines for histology. No significant correlations were found between Pfirrmann grade and mechanical data. However, polarized light microscopy images of disc sections indicated correlations between local tissue modulus measured with the TDI and the clarity and density of lamellar striations.

Effect of Ca2+ ions on the adhesion and mechanical properties of adsorbed layers of human osteopontin.

Using an atomic force microscope and a surface force apparatus, we measured the surface coverage, adhesion, and mechanical properties of layers of osteopontin (OPN), a phosphoprotein of the human bones, adsorbed on mica. OPN is believed to connect mineralized collagen fibrils of the bone in a matrix that dissipates energy, reducing the risk of fractures. Atomic force microscopy normal force measurements showed large adhesion and energy dissipation upon retraction of the tip, which were due to the breaking of the many OPN-OPN and OPN-mica bonds formed during tip-sample contact. The dissipated energy increased in the presence of Ca(2+) ions due to the formation of additional OPN-OPN and OPN-mica salt bridges between negative charges. The forces measured by surface force apparatus between two macroscopic mica surfaces were mainly repulsive and became hysteretic only in the presence of Ca(2+): adsorbed layers underwent an irreversible compaction during compression due to the formation of long-lived calcium salt bridges. This provides an energy storage mechanism, which is complementary to energy dissipation and may be equally relevant to bone recovery after yield. The prevalence of one mechanism or the other appears to depend on the confinement geometry, adsorption protocol, and loading-unloading rates.

Nanoscale ion mediated networks in bone: osteopontin can repeatedly dissipate large amounts of energy.

In the nanocomposite bone, inorganic material is combined with several types of organic molecules, and these complexes have been proposed to increase the bone strength. Here we report on a mechanism of how one of these components, human osteopontin, forms large mechanical networks that can repeatedly dissipate energy through work against entropy by breaking sacrificial bonds and stretching hidden length. The behavior of these in vitro networks is similar to that of organic components in bone, acting as an adhesive layer in between mineralized fibrils.

The role of calcium and magnesium in the concrete tubes of the sandcastle worm.

Sandcastle worms Phragmatopoma californica build mound-like reefs by sticking together large numbers of sand grains with cement secreted from the building organ. The cement consists of protein plus substantial amounts of calcium and magnesium, which are not invested in any mineral form. This study examined the effect of calcium and magnesium depletion on the structural and mechanical properties of the cement. Divalent ion removal by chelating with EDTA led to a partial collapse of cement architecture and cement dislodgement from silica surfaces. Mechanical properties examined were sand grain pull-out force, tube resistance to compression and cement adhesive force. EDTA treatment reduced sand grain pull-out forces by 60% and tube compressive strength by 50% relative to controls. EDTA lowered both the maximal adhesive force and energy dissipation of cement by up to an order of magnitude. The adhesiveness of calcium- and magnesium-depleted cement could not be restored by re-exposure to the ions. The results suggest that divalent ions play a complex and multifunctional role in maintaining the structure and stickiness of Phragmatopoma cement.